Characterization of murine TWEAK and its receptor (Fn14) by monoclonal antibodies

https://doi.org/10.1016/S0006-291X(03)01051-9Get rights and content

Abstract

In the present study, we characterized murine TWEAK and its receptor (Fn14) by generating cDNA transfectants and specific monoclonal antibodies (mAbs). Recombinant murine TWEAK bound to murine Fn14-transfected L5178Y (mFn14/L5178Y) cells and induced cell death. Some anti-human Fn14 mAbs we previously generated strongly cross-reacted with murine Fn14 and induced cell death in mFn14/L5178Y cells. Murine TWEAK-transfected L5178Y cells expressed murine TWEAK on cell surface and secreted functional TWEAK, which were detected by a newly generated anti-murine TWEAK mAb (MTW-1). Although thioglycolate-elicited murine peritoneal macrophages did not express a detectable level of TWEAK on their surface, they secreted functional TWEAK that was cytotoxic against mFn14/L5178Y cells and neutralized by MTW-1. The anti-murine TWEAK and Fn14 mAbs will be useful for further investigating the physiological and pathological functions of TWEAK and Fn14.

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Materials and methods

Cells. Mouse T lymphoma L5178Y and myeloma P3U1 (P3X63Ag8U.1) were cultured in RPMI 1640 containing 10% FCS, 100 μg/ml streptomycin and penicillin, and 2 mM glutamine (culture medium). L5178Y-derived transfectants, including mTWEAK/L5178Y and mFn14/L5178Y, were also maintained in the culture medium.

Reagents. mIFN-γ, butylated hydroxyanisole (BHA), and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) were purchased from BD Bioscience (San Diego, CA), Wako Pure Chemicals (Osaka, Japan),

Characterization of mFn14 transfectants

To characterize the functional properties of mouse Fn14 (mFn14), we first generated stable transfectants expressing full-length mFn14 cDNA. We used L5178Y cells for the transfection, since these cells did not bind CD8-mTWEAK as estimated by flow cytometry (Fig. 1A). The resulting mFn14/L5178Y cells exhibited a high CD8-mTWEAK binding (Fig. 1A). Recently, we have reported that CD8-hTWEAK could induce cell death in human Fn14-transfected L5178Y cells [10]. As shown in Fig. 1B, CD8-mTWEAK could

Acknowledgements

This work was supported by grants from the Human Frontier Science Program, the Ministry of Education, Science, Sports and Culture, and the Ministry of Health, Japan.

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